scholarly journals Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kazumasa Oda ◽  
Takashi Nomura ◽  
Takanori Nakane ◽  
Keitaro Yamashita ◽  
Keiichi Inoue ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Local Deformation ◽  
Proton Donor ◽  
Time Resolved ◽  
Molecular Events ◽  
Channel Opening ◽  
Ion Conducting ◽  
And Shifts ◽  
Opening And Closing

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

scholarly journals Photoactivation of Drosophila melanogaster cryptochrome through sequential conformational transitions

2019 ◽  
Vol 5 (7) ◽  
pp. eaaw1531 ◽  
Author(s):  
Oskar Berntsson ◽  
Ryan Rodriguez ◽  
Léocadie Henry ◽  
Matthijs R. Panman ◽  
Ashley J. Hughes ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structural Response ◽  
Biochemical Activity ◽  
Time Resolved ◽  
X Ray ◽  
Molecular Events ◽  
Carboxyl Terminal ◽  
Dynamics Simulations

Cryptochromes are blue-light photoreceptor proteins, which provide input to circadian clocks. The cryptochrome from Drosophila melanogaster (DmCry) modulates the degradation of Timeless and itself. It is unclear how light absorption by the chromophore and the subsequent redox reactions trigger these events. Here, we use nano- to millisecond time-resolved x-ray solution scattering to reveal the light-activated conformational changes in DmCry and the related (6-4) photolyase. DmCry undergoes a series of structural changes, culminating in the release of the carboxyl-terminal tail (CTT). The photolyase has a simpler structural response. We find that the CTT release in DmCry depends on pH. Mutation of a conserved histidine, important for the biochemical activity of DmCry, does not affect transduction of the structural signal to the CTT. Instead, molecular dynamics simulations suggest that it stabilizes the CTT in the resting-state conformation. Our structural photocycle unravels the first molecular events of signal transduction in an animal cryptochrome.

scholarly journals Light-induced Helix Movements in Channelrhodopsin-2

2014 ◽  
Vol 70 (a1) ◽  
pp. C1067-C1067
Author(s):  
Maria Mueller
Keyword(s):  
Conformational Changes ◽  
Transmembrane Helices ◽  
Dimer Interface ◽  
Channel Opening ◽  
Gated Channel ◽  
Ion Conducting ◽  
Lipid Environment ◽  
Opening And Closing ◽  
2D Crystals ◽  
State Difference

Electron crystallography has the unique advantage of visualizing membrane proteins in a native-like lipid environment, which likely favors the native conformation. In addition, it allows for the protein to undergo conformational changes in response to their activating signals. We used 2D crystals of channelrhodopsin-2, a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel et al., 2003) to study light-induced conformational changes of this intriguing channel, which is currently a powerful tool in optogenetics. Therefore, 2D crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6 Å resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) 2, 6, and 7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and 4 at the dimer interface. While conformational changes in TMH6 and 7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2.

scholarly journals Structural photoactivation of a full-length bacterial phytochrome

2016 ◽  
Vol 2 (8) ◽  
pp. e1600920 ◽  
Author(s):  
Alexander Björling ◽  
Oskar Berntsson ◽  
Heli Lehtivuori ◽  
Heikki Takala ◽  
Ashley J. Hughes ◽  
...  
Keyword(s):  
Time Scales ◽  
Conformational Changes ◽  
Structural Changes ◽  
Deinococcus Radiodurans ◽  
Full Length ◽  
Photon Absorption ◽  
Signaling Mechanism ◽  
Time Resolved ◽  
Binding Domains ◽  
Sensor Proteins

Phytochromes are light sensor proteins found in plants, bacteria, and fungi. They function by converting a photon absorption event into a conformational signal that propagates from the chromophore through the entire protein. However, the structure of the photoactivated state and the conformational changes that lead to it are not known. We report time-resolved x-ray scattering of the full-length phytochrome from Deinococcus radiodurans on micro- and millisecond time scales. We identify a twist of the histidine kinase output domains with respect to the chromophore-binding domains as the dominant change between the photoactivated and resting states. The time-resolved data further show that the structural changes up to the microsecond time scales are small and localized in the chromophore-binding domains. The global structural change occurs within a few milliseconds, coinciding with the formation of the spectroscopic meta-Rc state. Our findings establish key elements of the signaling mechanism of full-length bacterial phytochromes.

scholarly journals Photoactivation mechanism of a carotenoid-based photoreceptor

2017 ◽  
Vol 114 (24) ◽  
pp. 6286-6291 ◽  
Author(s):  
Sepalika Bandara ◽  
Zhong Ren ◽  
Lu Lu ◽  
Xiaoli Zeng ◽  
Heewhan Shin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Direct Interaction ◽  
Water Soluble ◽  
Site Directed Mutagenesis ◽  
Photochemical Property ◽  
Direct Observations ◽  
Absorbing State ◽  
Molecular Events ◽  
Orange Carotenoid Protein

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein–pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with site-directed mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto–enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.

scholarly journals Conformational changes in the catalytically inactive nucleotide-binding site of CFTR

2013 ◽  
Vol 142 (1) ◽  
pp. 61-73 ◽  
Author(s):  
László Csanády ◽  
Csaba Mihályi ◽  
Andras Szollosi ◽  
Beáta Töröcsik ◽  
Paola Vergani
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Nucleotide Binding ◽  
Structural Rearrangement ◽  
Nucleotide Binding Site ◽  
Binding Domains ◽  
Channel Opening ◽  
Catalytically Active ◽  
Opening And Closing ◽  
Closing Rate

A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C→O1→O2→C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N6-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2×) transition O1→O2 and decreases the nonhydrolytic closing rate (transition O1→C) of CFTR mutants K1250A (∼4×) and E1371S (∼3×). Mutation H1348A also slowed (∼3×) the O1→O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A (∼3×) and E1371S (∼3×) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1→C and O1→O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

scholarly journals Retinal isomerization and water-pore formation in channelrhodopsin-2

2018 ◽  
Vol 115 (14) ◽  
pp. 3557-3562 ◽  
Author(s):  
Albert Ardevol ◽  
Gerhard Hummer
Keyword(s):  
Schiff Base ◽  
Conformational Changes ◽  
Pore Formation ◽  
Proton Acceptor ◽  
Time Resolved ◽  
Channel Opening ◽  
Selective Channel ◽  
Extracellular Side ◽  
Membrane Spanning ◽  
Time Resolved Infrared Spectroscopy

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P2390–like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.

scholarly journals Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

2021 ◽  
Author(s):  
Toshiaki Hosaka ◽  
Takashi Nomura ◽  
Minoru Kubo ◽  
Takanori Nakane ◽  
Luo Fangjia ◽  
...  
Keyword(s):  
Ion Transport ◽  
Conformational Changes ◽  
Structural Changes ◽  
Ion Transfer ◽  
Ion Release ◽  
Time Resolved ◽  
Iodide Ions ◽  
Lateral Displacements ◽  
Extracellular Side ◽  
Cytoplasmic Side

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

scholarly journals Photocage-initiated time-resolved solution X-ray scattering investigation of protein dimerization

IUCrJ ◽  
2018 ◽  
Vol 5 (6) ◽  
pp. 667-672 ◽  
Author(s):  
Inokentijs Josts ◽  
Stephan Niebling ◽  
Yunyun Gao ◽  
Matteo Levantino ◽  
Henning Tidow ◽  
...  
Keyword(s):  
Small Molecule ◽  
Conformational Changes ◽  
Structural Changes ◽  
Time Resolved ◽  
Protein Dimerization ◽  
X Ray ◽  
Single Turnover ◽  
X Ray Scattering ◽  
Photoactivatable Proteins ◽  
Ray Scattering

This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated time-resolved X-ray solution scattering by observing the ATP-driven dimerization of the MsbA nucleotide-binding domain. Photocaged small molecules allow the observation of single-turnover reactions of non-naturally photoactivatable proteins. The kinetics of the reaction can be derived from changes in X-ray scattering associated with ATP-binding and subsequent dimerization. This method can be expanded to any small-molecule-driven protein reaction with conformational changes traceable by X-ray scattering where the small molecule can be photocaged.

Conformational Changes in BK(Ca) Channel during Channel Opening and Closing Revealed by Optical Methods

2002 ◽  
Vol 96 (Sup 2) ◽  
pp. A770
Author(s):  
Riccardo Olcese ◽  
Ning Zhu ◽  
Trude Haug ◽  
Ligia Toro ◽  
Enrico Stefani
Keyword(s):  
Conformational Changes ◽  
Optical Methods ◽  
Ca Channel ◽  
Channel Opening ◽  
Opening And Closing

scholarly journals Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls

2017 ◽  
Vol 114 (52) ◽  
pp. 13708-13713 ◽  
Author(s):  
Yingyuan Sun ◽  
Aaron P. Roznowski ◽  
Joshua M. Tokuda ◽  
Thomas Klose ◽  
Alexander Mauney ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cell Walls ◽  
Structural Changes ◽  
The Other ◽  
Host Bacterium ◽  
Time Resolved ◽  
Viral Attachment ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.

Sign in / Sign up

Export Citation Format

Share Document